Compositions, methods, and computer systems related to making and administering modified t cells

ABSTRACT

Embodiments described herein relate to methods, devices, and computer systems thereof for the derivation of T CAR libraries (Universal Subject or Individual Subject) for personalized treatment of disease in a subject. In certain embodiments, differential screening of normal and diseased tissue expression data is utilized to determine disease-specific antigens and thereby generate T CAR cells reactive to such antigens to form a disease-specific library. In certain embodiments, determination of the most effective T CAR clones from the disease-specific library is based on the subject&#39;s own disease-specific antigens. In certain embodiments, a subject is treated with a therapeutically effective amount of T CAR clones.

If an Application Data Sheet (ADS) has been filed on the filing date ofthis application, it is incorporated by reference herein. Anyapplications claimed on the ADS for priority under 35 U.S.C. §§119, 120,121, or 365(c), and any and all parent, grandparent, great-grandparent,etc. applications of such applications, are also incorporated byreference, including any priority claims made in those applications andany material incorporated by reference, to the extent such subjectmatter is not inconsistent herewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and/or claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Priority Applications”), if any, listed below(e.g., claims earliest available priority dates for other thanprovisional patent applications or claims benefits under 35 USC §119(e)for provisional patent applications, for any and all parent,grandparent, great-grandparent, etc. applications of the PriorityApplication(s)). In addition, the present application is related to the“Related Applications,” if any, listed below.

PRIORITY APPLICATIONS

None.

RELATED APPLICATIONS

United States patent application No. To be Assigned, entitledCOMPOSITIONS, METHODS, AND COMPUTER SYSTEMS RELATED TO MAKING ANDADMINISTERING MODIFIED T CELLS, naming Roderick A. Hyde, Wayne R.Kindsvogel and Gary L. McKnight as inventors, filed 14 Mar. 2013 withattorney docket no. 0412-004-001-000000, is related to the presentapplication.

United States patent application No. To be Assigned, entitledCOMPOSITIONS, METHODS, AND COMPUTER SYSTEMS RELATED TO MAKING ANDADMINISTERING MODIFIED T CELLS, naming Roderick A. Hyde, Wayne R.Kindsvogel and Gary L. McKnight as inventors, filed 14 Mar. 2013 withattorney docket no. 0412-004-001A-000000, is related to the presentapplication.

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation, continuation-in-part, or divisional of a parentapplication. Stephen G. Kunin, Benefit of Prior-Filed Application, USPTOOfficial Gazette Mar. 18, 2003. The USPTO further has provided forms forthe Application Data Sheet which allow automatic loading ofbibliographic data but which require identification of each applicationas a continuation, continuation-in-part, or divisional of a parentapplication. The present Applicant Entity (hereinafter “Applicant”) hasprovided above a specific reference to the application(s) from whichpriority is being claimed as recited by statute. Applicant understandsthat the statute is unambiguous in its specific reference language anddoes not require either a serial number or any characterization, such as“continuation” or “continuation-in-part,” for claiming priority to U.S.patent applications. Notwithstanding the foregoing, Applicantunderstands that the USPTO's computer programs have certain data entryrequirements, and hence Applicant has provided designation(s) of arelationship between the present application and its parentapplication(s) as set forth above and in any ADS filed in thisapplication, but expressly points out that such designation(s) are notto be construed in any way as any type of commentary and/or admission asto whether or not the present application contains any new matter inaddition to the matter of its parent application(s).

If the listings of applications provided above are inconsistent with thelistings provided via an ADS, it is the intent of the Applicant to claimpriority to each application that appears in the Priority Applicationssection of the ADS and to each application that appears in the PriorityApplications section of this application.

All subject matter of the Priority Applications and the RelatedApplications and of any and all parent, grandparent, great-grandparent,etc. applications of the Priority Applications and the RelatedApplications, including any priority claims, is incorporated herein byreference to the extent such subject matter is not inconsistentherewith.

SUMMARY

Various embodiments described herein relate to compositions, methods,and computer systems for constructing T cell clone(s) exhibiting anartificial Chimeric Antigen Receptor(s) as part of a library. Variousembodiments described herein relate to administering the T cell clonecell line(s) from the library to a subject in need thereof.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a partial view of an embodiment related to engineering anartificial T cell line.

FIG. 1B is a partial view of an embodiment related to engineering anartificial T cell line.

FIG. 2A is a partial view of an embodiment related to constructing amulti-specific chimeric antigen receptor.

FIG. 2B is a partial view of an embodiment related to constructing amulti-specific chimeric antigen receptor.

FIG. 2C is a partial view of an embodiment related to constructing amulti-specific chimeric antigen receptor.

FIG. 2D is a partial view of an embodiment related to constructing amulti-specific chimeric antigen receptor.

FIG. 3 is a partial view of an embodiment related to engineering anartificial T cell line, and computer systems thereof.

FIG. 4 is a partial view of an embodiment related to engineering anartificial T cell line, and computer systems thereof.

FIG. 5 is a partial view of an embodiment related to engineering anartificial T cell line and computer systems thereof.

FIG. 6 is a partial view of an embodiment related to engineering anartificial T cell line and computer systems thereof.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

Described herein are various embodiments related to Universal SubjectDisease Specific Library of cytolytic T CAR cells (T cells withartificial Chimeric Antigen Receptors) and Individual Subject DiseaseSpecific Library of cytolytic T CAR cells. In an embodiment, the T CARcells represent a universal T cell clone that has been manipulated toartificially and specifically recognize a particular target antigen(e.g., a disease specific antigen). In an embodiment, the T CAR cellsprovide a tool for immunotherapy of a subject afflicted with a diseaseor disorder. Also described herein are computer systems utilized inconjunction with the methods of making or administering the T CAR celllibraries to a subject.

Artificial Cytolytic T Cell Clone Universal Subject Disease SpecificLibrary

In an embodiment, a Universal Subject Disease Specific Library ofartificial cytolytic T cells is developed that bear a multi-specificartificial chimeric antigen receptor (CAR), as described in detailherein. In an embodiment, the development of a multi-specific T cell CARclone Universal Subject Disease Specific Library provides anHLA-independent recognition of antigen. In an embodiment, the libraryprovides a rapid and effective treatment of immune-related disorders anddiseases. In an embodiment, the library is utilized to make a diseasespecific therapeutic T cell line. Such CAR is engineered, for example,by using available sequence data or expression data derived frompopulation studies of multiple tissue types from multiple subjects. Fromthis data, in an embodiment antigens are identified from all presentlyidentified cell surface antigens and secreted proteins for a particularspecies of subject (e.g., human), or from all identified cell surfaceantigens and secreted proteins for a specific tissue type (e.g. breastcancer tissue), based on tumor or diseased tissue vs. normal tissue.This differential expression screen identifies antigens specificallyassociated with various tissues, of either normal or diseased state.

In an embodiment, the expression data are determined by screeningprimary tumor tissue against normal tissue. In an embodiment, theexpression data are determined by screening primary tumor tissue againstsecondary tumor tissue. In an embodiment, the expression data aredetermined by screening primary tumor tissue or secondary tumor tissueagainst metastasized tissue.

In an embodiment, a subject includes a mammal, bird, fish, reptile, oramphibian. In an embodiment, a subject includes a vertebrate orinvertebrate. In an embodiment, a subject includes a plant. In anembodiment, a subject includes a human. In an embodiment, the subjectincludes a fetus in utero.

In an embodiment, the genetic expression data are derived from multipletumors from many subjects in order to provide an array of diseasespecific antigens. Thus, in an embodiment, multiple disease sites frommultiple diseased subjects are utilized for sampling of geneticexpression data, or multiple cell samples from a diseased subject. Forexample, in an embodiment, the expression data are derived from multipledisease sites (e.g., joint synovial fluid and/or cell samples) frommultiple subjects afflicted with rheumatoid arthritis or systemic lupuserthematosus. In an embodiment, the expression data are derived frommultiple central nervous system (e.g., spinal tap) fluid and/or cellsamples, from multiple subjects afflicted with multiple sclerosis. In anembodiment, the expression data are derived from multiple colonbiopsies, from multiple subjects afflicted with Crohn's disease orInflammatory Bowel Disease. In an embodiment, the expression data arederived from multiple sputum or saliva samples, from multiple subjectsafflicted with asthma. In an embodiment, the expression data are derivedfrom multiple blood samples, from multiple subjects afflicted withdiabetes, Graves' disease, Hashimoto's thyroiditis, Myasthenia gravis,vasculitis, or other immune related disorder. These are non-limitingexamples of tissues that may be tested for comparison with normal tissuefrom either the same subject or from a healthy subject not afflictedwith the same disease or disorder as the testing subject. It isunderstood that various tissues (including lymph, blood, saliva, urine,or other bodily fluid and/or cell sample) may be utilized for testingfor cell surface expression of antigens and secreted proteins related toa particular disease or disorder.

In an embodiment, a library of T cell CAR clones is constructed for eachparticular tissue type or state of diseased tissue type. For example, alibrary can be constructed for primary tumors in breast cancer, aseparate library can be constructed for secondary tumors in breastcancer, and a separate library can be constructed for metastatic breastcancer. In another example, a library can be constructed for multiplesclerosis, a separate library for Alzheimer's disease. In anotherexample, a library can be constructed for early stage Alzheimer'sdisease, and a separate library constructed for later state Alzheimer'sdisease. It is understood that these are non-limiting examples and canreadily be applied to other tissue types or diseases.

In an embodiment, the tissue type for purposes described herein includesbut is not limited to, breast tissue, prostate tissue, colon tissue,stomach tissue or other gastro-intestinal tissue, uterine tissue, eyetissue, ear tissue, skin tissue, blood, nasal tissue, mouth or throattissue (such as oral mucosa, pharyngeal or laryngeal tissue, tongue)joint tissue, bone tissue, bone marrow tissue, scalp tissue, muscletissue, ovarian tissue, testicular tissue, or fetal tissue.

In an embodiment, a computer algorithm is used to prioritize the mostcommonly expressed cell surface antigens for targeting in theengineering of the artificial T CAR cell clones or the spatial ortemporal expression of one or more antigens. The T CAR cell clones areutilized for adoptive T cell therapy as a treatment modality forimmunotherapy for tumor regression in cancer, or other immunologicalillnesses or afflictions. However, if a T CAR library is generated witheach CAR recognizing only one antigen (with or without recognizingmultiple epitopes of the only one antigen), then the library is“universal,” and can be utilized for any human disease. In this case, acomputer algorithm is used to prioritize the most commonly expressedcell surface antigens for a particular disease or cell/tissue type.

For example, a computing device or computing system running an algorithmor computer program is used to select the top several most-commonlyexpressed cell surface antigens and secreted proteins specified for aparticular target tissue. The cell surface antigens and secretedproteins can be determined by FACS, gene microarray, RT-PCR, etc. orother standard techniques. In another example, a computer algorithm canbe used to select antigens based on their structural formation,post-translational modification, Kd value based on previously identifiedantibodies, genetic sequence, mRNA sequence, peptide sequence, or othercharacteristic(s).

Many computer software analysis algorithms and programs have beendeveloped to analyze expression data. For example, the expression datacan be analyzed with Significance analysis of microarrays (SAM)developed by Stanford, ANOVA, ANOSVA (for analysis of splice variation),Bonferroni, Bayesian probability, Markov chains, Monte Carlo methods, orGaussian distributions. See, for example, Baldi, et al. Bioinformatics,vol. 17, no. 6, 2001, pp. 509-519, which is incorporated herein byreference.

Based on the analysis of the information of the population diseasedtissue expression studies, a multi-specific T CAR cell clone library isconstructed as described below. For example, targeting of specific tumorspecific antigen (TSA) groups or subgroups can be correlated byanalyzing expression studies of genomic and transcription changes. Forexample, inherited variants (copy number variants and single nucleotidepolymorphisms) and acquired somatic copy number aberrations (CNAs) havebeen shown to be associated with expression in roughly 40% of genes,with the dominant being cis- and trans-acting CNAs. See Curtis, et al.Nature, vol. 486, pp. 346-352, 2012, which is incorporated herein byreference.

In an embodiment, a published genomic listing is utilized thatidentifies all presently identified cell surface antigens on humancells. See, for example, Cunha, et al. PNAS, 106: 16752-16757 (2009),which is incorporated herein by reference. In an embodiment, utilizingthis genetic sequence listing of cell surface antigens, T CAR cells areconstructed with single chain antibodies providing specific binding toone or more cell surface antigens on human cells. For example, singlechain variable region fragments (scFvs) which specifically bind to asingle cell surface antigen from the listing are identified, forexample, by screening phage display libraries comprised of human scFv.More details are included in Example 3 herein. Thus, in an embodiment,multi-specific T CAR cells are engineered for each of the presentlyidentified human cell surface antigens (approximately 3700 proteins),and optionally various combinations thereof. For example, in anembodiment, a T CAR recognizes only one antigen. In an embodiment, a TCAR recognizes multiple antigens.

Likewise, in an embodiment, a series of Western blots is conductedbetween normal tissue and diseased tissue from multiple subjects, orarrays, RT-PCR (optionally quantitative real-time, etc.), and othertechniques are utilized for detecting glycosylation orpost-translational modifications. In this way, in an embodiment, thetarget antigens are identified based on differences in genetic sequence,mRNA sequence, or post-translational modification (e.g., glycosylation),and from the identified target antigens, T CAR cells are constructed. Inan embodiment, mRNA isolated from a disease site in a subject issequenced and the sequence information compared by computer system withthe identified sequences. In an embodiment, such a differentialscreening out of the normal sequences and identification of abnormalgene expression sequences is utilized to identify the target antigensfor the disease of the subject.

It is understood that an antigen, as recognized by one of skill in theart, includes a substance capable of the lock-and-key interaction of onemolecule (the antigen) with another (the antibody or lymphocytereceptor). It is further understood that an antigen includes peptides,glycopeptides, peptidoglycans, lipids, proteoglycans, glycoproteins,glycolipids, and the like. It is understood that antigen specificityincludes the ability of a lymphocyte (e.g., T CAR cell) to recognize anantigen specifically as a unique molecule and discern it from anotherantigen with a high level of precision. Antigen specificity isattributed to the primary, secondary, and tertiary structure, includingside-chain conformations of the antigen. Moreover, an epitope includesthe portion of the antigen that provides the recognition of the antigenby the lymphocyte receptor (e.g., T CAR cell).

Next, based on the selected antigens derived from the expression datadescribed above, an array of single chain variable fragments (scFv) areidentified through the use of identified antibodies against theparticular antigens, or by way of screening the scFvs for the antigen(s)if antibodies are not yet readily available. In an embodiment, theselection of scFvs is conducted for example, by way of phage display, asdescribed herein. In an embodiment, the selection of scFvs is conductedby way of ribosome display. See, for example, Lee et al. JIM, 284 (2004)147-157, which is incorporated herein by reference. In an embodiment theselection of scFvs is conducted by way of expression in transgenicorganism (e.g., plant or animal).

Therefore, in an embodiment, regardless of how the sequences of targetantigens are derived, once such sequences have been obtained, a pair ofscFvs will be developed for each pair of antigens and the scFvs will beincorporated into a CAR, an artificial T cell Chimeric Antigen Receptor.The corresponding sequences are converted into a retroviral (e.g.,lentiviral) vector, for example, according to standardized techniques.In an embodiment, the expression vector(s) and utilized to infect Tcells, thus resulting in a universal T cell clone that has beenmanipulated to artificially and specifically recognize a particulartarget antigen. The summation of all such target-specific T CAR cellclones comprises the T CAR cell library of clones.

In an embodiment, each T cell clone bears a multi-specific CAR thatelicits a polyclonal response by targeting multiple epitopes of the sameantigen, which is usually much stronger than a monoclonal reactionbetween a T cell and an antigen. In addition, the polyclonal response ofthe T CAR cell may be more efficacious in a clinical setting. (See, forexample, Phuphanich, et al. Cancer Immunol. 62: 125-135, 2013, which isincorporated herein by reference).

In an embodiment, the T cell CAR is engineered to include two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, or ten or more target sequence specificbinding sites. This multi-specific CAR provides a higher level ofspecificity, and thus increased binding strength, for the target tissue.In an embodiment, the data expression analysis determines a distinctiveantigen profile for a specific tissue disease or disease state. Forexample, utilizing heterogeneous expression profile analysis, it wasfound that among tumor-specific antigens, serum autoantibodies againstseven candidate targets were detected in 4-11% of patients with lung andstomach cancers. See, for example, Xu et al. Cancer Res. 72(24):1-11(2012), which is incorporated herein by reference. A similar algorithmapplied in Xu is adapted for use in certain embodiments describedherein. In an embodiment, coupled two-way clustering analysis of theexpression data is employed. See, for example, Getz et al. PNAS 97; 22(2000) 12079-12084, which is incorporated herein by reference. In anembodiment, a web-browser interface based database such as ArrayDB, isutilized for expression data analysis. See for example, Ermolaeva, etal. Nat. Genet. 1998 (20)1: 19-23 (1998), which is incorporated hereinby reference.

Thus, in an embodiment, for the Universal Subject Disease SpecificLibrary, a differential screen is performed by comparing cell expressiondata of diseased cells (e.g., tumor cells) from multiple cell samples(e.g., multiple tissues or organs) of multiple subjects, with normaltissue from multiple cell samples of multiple subjects. The parametersfor normal tissue expression can be adjusted for example, in order toreduce background noise or fine-tuned in order to deduce a truedifferential expression for a particular target antigen.

The resulting data from this differential screen is manipulated by acomputer (based on quantity of expression of antigen(s), quality ofexpression of antigen(s), Kd of binding of antigen(s) to cognatereceptor(s) or antibody, or other characteristics) and derives theUniversal Subject Disease Specific Antigens (UDSA) for a particulardisease or disorder. In an embodiment, The UDSA are measured orcategorized quantitatively and/or qualitatively. That is, in anembodiment, the UDSA and gene expression information related thereto arecataloged or organized based on the relative quantity of expression inthe diseased tissue (e.g., the quantity of antigens, n=about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 30, about 40, about 50, or any valuetherebetween), and/or based on the quality of the expression in thediseased tissue (e.g., genetic or mRNA sequence, glycosylation or otherpost-translation modifications, etc.). In an embodiment, the UDSA andgene expression information related thereto are cataloged or organizedbased on one or more of a genetic or mRNA mutation, abnormalpost-translational modification, Kd value that varies forantigen-receptor binding, methylation/acetylation of nucleic acids,lipid modifications, or other structural characteristics. Thus, the geneexpression information includes any of the qualitative or quantitativecharacteristics disclosed herein.an

For example, glycosylation or other post-translational modifications areanalyzed by way of sequencing, linkage analysis (e.g., enzymaticcleavage or otherwise), carbohydrate separation methods (e.g.,high-performance liquid chromatography), and other means. Likewise,genetic or mRNA sequence aberrations or mutations can be detected bysequencing, enzymatic cleavage, or other standard techniques.

In an embodiment, the UDSA and gene expression information relatedthereto are cataloged or organized based on one or more of the totalnumber of antigens expressed in a diseased tissue versus normal tissue,the relative number of antigens expressed in a diseased tissue versusnormal tissue, a cluster expression of several particular antigens in adiseased tissue versus normal tissue, the absence of expression of anormal tissue antigen in a diseased tissue, cell surface density of asingle antigen, cell surface concentration of a single antigen, ornumber of copies of an antigen per cell.

The UDSA are then utilized to make a Universal Subject Disease SpecificLibrary of multi-specific CARs that recognize disease specific antigensfound most commonly in the general population of subjects. Details ofthe molecular biology of making the Universal Subject Disease SpecificLibrary are discussed further herein. Once the Universal Subject DiseaseSpecific Library has been generated, a subject afflicted with theparticular disease for which the UDSAs have been identified has its owndiseased cell samples analyzed for cell expression. Based on the resultsof the subject's own diseased cell sample analysis, a computer system isutilized to select multi-specific clones from the Universal SubjectDisease Specific Library that match the subject's own diseased cellexpression profile. In this way, the subject receives multi-specificclones designed specifically for its individual treatment based on itsown diseased cell sample expression data. In an embodiment, multiplelibraries are generated each based on various stages of disease (e.g.,progressive chronic diseases, metastatic cancer, etc.) compared withnormal tissue or other disease states of the tissue.

By contrast, in an embodiment, for the Individual Subject DiseaseSpecific Library, a differential screen is performed by comparing cellexpression data of the subject's own diseased cells (e.g., tumor cells),with normal tissue from the same subject. In an embodiment, if multipletissues or organs are afflicted (e.g., metastasis of a tumor), then eachtissue or organ is included in separate expression analysis or the sameexpression analysis, depending on the desired treatment regimen. Forexample, in a subject with no histologically detected metastasis of theprimary tumor or disease site, the expression analysis is conducted withthe primary tumor vs. normal tissue antigen profile. In an embodiment,the expression analysis includes the individual's specific diseaseanalysis of primary tumor vs. normal tissue and further includesanalysis to select antigens from a universal metastasis antigen profile,even if no metastases is present or detected in this particular subject.In that way, a prophylactic treatment to target metastases is conductedin conjunction with the specific primary disease antigen profile. Inanother example, the expression analysis includes the individual'sprimary disease site antigen profile as well as secondary disease sites(e.g., metastases). In this way, multiple targets of disease areattacked efficiently and effectively.

In an embodiment, a multi-specific CAR specifically recognizes two ormore epitopes of the same antigen. In an embodiment, the physicalproximity of the multi-specific CAR that specifically recognizes two ormore epitopes of the same antigen provides a benefit in that thelikelihood of receptor binding is increased and clustering of multipleseparate antigen receptors at the T cell surface is not needed since themulti-specific receptors are physically located on the same CAR.

In an embodiment, a computing device or computing system running analgorithm or computer program is used to analyze the Kd bindingstrengths of the multi-specific CAR, for example, by analysis of thebinding strength of each selected antigen binding site with its targetsequence, as well as the coordinated binding strength of the entiremulti-specific receptor. For example, in certain CARs the spatialpattern of the multiple specific target sites is optimized based on thesequence in which the specific antigen sites occur in the CAR, or basedon the spatial binding relationship with the particular targets. In anembodiment, the multi-specific CAR provides at least 2 or more, at least3 or more, at least 4 or more, at least 5 or more, at least 6 or moreantigen binding sites (indicating receptor specificity) (e.g., scFvs).In an embodiment, one or more multi-specific CARs are included on asingle T cell clone. In an embodiment, two or more, three or more, orfour or more multi-specific CARs are included on a single T cell clone.

In an embodiment, the T cell clone is constructed by assembling thespecific nucleotide sequences selected by, for example, splice overlapPCR and standard cloning methods. See, for example, U.S. Pat. No.6,410,319, which is incorporated herein by reference. Utilizing standardcloning techniques (e.g., RT-PCR), ribosome binding sequences, signalpeptides (e.g., OX40 or 41BB), and the particular sequences selected fordetermining the specificity of targeting for that individual clone aswell as any desired linkers, are spliced and joined together into afinal genetic construct that is then cloned into a mammalian expressionvector (e.g., pcDNAneo or similar vector, under the control of a CMVimmediate-early or similar promoter). Proper assembly of the artificialconstruct is confirmed, for example, by DNA sequence analysis of thefinal product.

In an embodiment, T cells are then transfected with the artificial CARconstruct according to standard techniques, and are analyzed (e.g., byFACS and/or Western Blot and/or Fluorescence in situ hybridization(FISH)) for confirmation that the cells harbor the non-naturallyoccurring construct.

In an embodiment, T cells expressing the multi-specific CAR are testedfor in vitro stimulation of cytokine production, for cytolytic activity,and (where appropriate) induction of any apoptotic or “suicide” genesoptionally included in the artificial CAR construct. For example, in anembodiment, the T cell clone includes a conditionally inducible suicidegene, such as thymidylate kinase, inducible caspase 9, CD20, thymidinekinase, or modified FAS. Such in vitro testing is done according tostandard techniques, for example, ELISA assays for IL-2 can be utilizedto test for in vitro stimulation of cytokine production (IL-2 iscommonly tested since it induces cell proliferation among lymphocytes).Chromium Release Assays can be used for measuring the cytolytic activityof the clones by allowing a target cell to take up radioactivesupernatant, followed by washing residual radioactivity away andincubating with the T cell clone presumed to be cytolytic. Measurementof release of the radioactivity indicates killing of the cells by the Tcell clone. In vitro cell proliferation assays utilized to measure theclones' ability to proliferate themselves can also be measured bystandard techniques, including, for example, my measuring tritiatedthymidine (thymidine is incorporated into the DNA of dividing cells).See U.S. Pat. No. 6,410,319, incorporated herein by reference, for moredetails on some of these standardized techniques.

A subject afflicted by or having symptoms of a disease or disorder, forexample an immune-related disease or disorder (such as cancer) istreated with the appropriate multi-specific T cell clone(s) selectedfrom the Universal Subject Disease Specific Library based on analysis ofa cell sample (e.g., biopsy) the subject's specifically selected targetantigens (such as tumor specific antigens) identified by use of amicroarray, FACS, or other standard technique. In this manner, themulti-specific T cell clone Universal Subject Disease Specific Libraryis analyzed (optionally by computing device or computing system runningan algorithm to identify the optimal T cell clone based on informationdetermined from expression studies of the subject's tissue (e.g., tumortissue)) and the optimal T cell clones are selected for the subject. Inan embodiment, the subject is treated by infusing one or moretherapeutically effective dose of the cytolytic T cell clones, eachexpressing the particular multi-specific artificial CAR.

In an embodiment, prior to, during, or subsequent to infusing thesubject with the first cohort of T cell CAR clones, a sample of cells(e.g., from a biopsy) from the subject's tumor or other target tissue isextracted and tested for cytolysis with the selected T cell CAR clonesexpected to be used for therapy. In this way, the relative cytolysis isdetermined, allowing for a more accurate dosing range and schedule forthe subject's therapy without causing undue cytotoxicity. Such dosingand scheduling are performed as for any standard clinical trials ortesting, and may include one or more parameters specific to theindividual test subject.

Artificial Cytolytic T Cell Clone Individual Subject Disease SpecificLibrary

In an embodiment, an Individual Subject Disease Specific Library ofcytolytic T cells expressing an artificial chimeric antigen receptor(CAR), as described in detail herein, is personalized specifically to anindividual subject's own disease (e.g., tumor) antigen expression dataor expression patterns, rather than population expression studies. Forexample, by screening the Universal Subject Disease Specific Library fora particular tissue or disease with an individual subject's diseasedtissue, antigens specific for the individual subject's diseased tissueare identified and isolated. In an embodiment, this information providesthe basis for determining and/or constructing appropriate T CAR cellclones for therapeutic administration to the individual subject. In anembodiment, a database of previously identified antigen sequences isscreened with the Individual Subject's diseased tissue and/or sequencestherefrom in order to determine the specific disease antigens associatedwith the Individual Subject's disease.

In an embodiment, the disease specific library is utilized to generate adisease specific therapeutic T cell line. Such CAR is engineered, forexample, as described herein for the Universal Subject Disease SpecificLibrary, except that in order to generate the CAR for the IndividualSubject Disease Specific Library, sample cells (e.g., from a biopsy)from the subject afflicted with cancer or another immune disease aresubjected to, for example, Next Generation Sequencing (NGS) which is astandard technique that provides a fast, high-quality sequence of themRNA transcriptome of the sample cells. See, for example, Mortazavi, etal., Nature Methods 5:621-628, 2008, which is incorporated herein byreference). For example, a differential screening of tumor vs. normalcell mRNA can be conducted for an individual subject with specificdisease. The standard techniques of constructing the T CAR cell clonesand library described herein for the Universal Subject library can beutilized for the Individual Subject Library.

As similarly described for the Universal Subject Disease SpecificLibrary, in an embodiment, a computer algorithm is used to prioritizethe most commonly expressed cell surface antigens and secreted proteinsfor targeting in the engineering of the artificial cytolytic T CARcells, or the spatial or temporal expression of one or more antigens(e.g., Protease-activated receptors (PAR receptors) are a Gprotein-coupled receptors that are activated by cleavage of part oftheir extracellular domain). The T cell CAR clones are utilized foradoptive T cell therapy as a treatment modality for immunotherapy fortumor regression in cancer, or other immunological illnesses orafflictions. The remainder of the process of constructing and utilizingthe cytolytic T cell CAR clones is carried out in the same manner asdescribed for the Universal Subject Disease Specific Library herein.

Based on the analysis of the mRNA transcriptome information of thesubject's tumor, a multi-specific T cell CAR clone library isconstructed in a similar manner as described for the Universal SubjectDisease Specific Library herein.

The resulting data from this differential screen is manipulated by acomputer (based on quantity of expression of antigen(s), quality ofexpression of antigen(s), Kd of binding of antigen(s) to cognatereceptor(s) or antibody, or other characteristics) and derives theIndividual Disease Specific Antigens (IDSA) for the individual subject'sparticular disease or disorder. In an embodiment, where an IndividualSubject is afflicted with multiple diseases simultaneously orsequentially, one or more Individual Subject Disease Specific Librariesare generated—each for use with one particular disease or disorder.Thus, if a subject is afflicted with multiple sclerosis, and latercontracts kidney cancer, that subject can be treated with clones from alibrary specific for the subject's multiple sclerosis, and later for thesubject's kidney cancer. In an embodiment, the IDSA are measured orcategorized quantitatively and/or qualitatively, similarly as describedherein for the Universal Subject Disease Specific Library.

The IDSA are then utilized to make an Individual Subject DiseaseSpecific Library of multi-specific CARs to disease specific antigensfound most commonly in the subject's own diseased cells. Details of themolecular biology of making the Individual Subject Disease SpecificLibrary are discussed further herein. Once the Individual SubjectDisease Specific Library has been generated, a computer system isutilized to select multi-specific clones from the Library for use invarious treatment sequences or dosages. In this way, the subjectreceives customized treatment for its own cell expression based on itsdiseased cells.

Construction of Multi-Specific CAR for Either Universal Library orIndividual Subject Library

In an embodiment, the multi-specific CAR is genetically engineered as anartificial T cell receptor specifically targeting the computer-selectedIDSA or UDSA. For example, in an embodiment, the CAR is a cell surfacemolecule including one or more extracellular domain (the specifictargeting region of the receptor), one or more intracellular signalingdomain, and one or more transmembrane domain.

In an embodiment, the CAR genetically disrupts the innate T cellreceptor of the engineered cell. In an embodiment, the geneticdisruption of the innate T cell receptor results in the completeelimination of the innate T cell receptor function. This elimination ofinnate T cell receptor function operates to prevent allogeneic response(e.g., graft vs. host disease) by the T CAR cells on the subjectreceiving them during therapy.

In an embodiment, individual components of the T CARs are produced fromnucleic acid molecules using standard molecular biology methods. Forexample, as described herein, nucleic acid molecules are inserted into avector that is able to express the fusion polypeptide when introducedinto an appropriate host cell (e.g., bacteria, yeast, insect, mammaliancells, or other cells). In an embodiment, any of the many standardtechnical methods can be used for insertion of DNA fragments into avector, as well as for constructing an expression vector encoding thefusion polypeptides described herein and under the control oftranscriptional or translational control signals. For example, one ormore of these methods may be used for in vitro recombinant DNA orsynthetic techniques or in vivo recombination.

In an embodiment, expression of the fusion polypeptides may be regulatedby a second nucleic acid sequence wherein the polypeptide(s) isexpressed in a host transformed with the recombinant DNA molecule (i.e.,expression of the polypeptide may be controlled by any promoter/enhancerelement utilized for standard practice.)

In an embodiment, the individual T cell clones are deficient in at leastone Human Leukocyte Antigen (HLA), including for example, HLA I-, HLAII-, HLA E or HLA G+. For example, the HLA I-HLA II- is engineered bydeleting the innate T cell receptor alpha region in beta2M (HLA classI-), and in HLA DR, HLA DQ or HLA DP regions (for HLA class II-).Standard methods of gene inactivation that can be utilized for thisparticular component include enforced methylation, siRNA, or shRNA. TheHLA I- and HLA II-conditions operates to prevent host T cell eliminationof the engineered T cell clone, whereas the forced expression of HLA Eor HLA G operates to prevent NK cell elimination of the engineered Tcells once administered to a subject.

In an embodiment, individual T cells used for generating T CAR cells canbe CD4+/CD8−, CD4−/CD8+, CD4−/CD8−, or CD4+/CD8+. In an embodiment, theT cells can be a mixed population of CD4+ and CD8+ cells, or can be apopulation propagated from a single clone. In an embodiment, the T cellsare a mixed population of CD4+ cells that produce IL-2 or othercytokines when their receptor engages the specific IDSA or UDSA, andCD8+ cells that lyse the target cells when their receptor engages thespecific IDSA or UDSA. In an embodiment, T cells that have been modifiedas described herein are referred to as T CAR cells. In an embodiment,the T cell includes a memory T cell with a phenotype of CD62L+ CD45RA−.

In an embodiment, the individual T cells are primary T CAR cells, suchas from peripheral blood mononuclear cells (PBMC). In an embodiment, theindividual T CAR cells are engineered from stem cells from the bonemarrow, peripheral blood, hepatic, spleen, or other stem cellcompartment. In an embodiment, the T CAR cells are engineered fromhematopoietic stem cells. In an embodiment, the T cell CAR clones areexpanded prior to or subsequent to differentiation into T cells.

In an embodiment, the target antigen(s) described herein include one ormore isolated nucleic acid molecule(s). In an embodiment, the one ormore isolated nucleic acid molecules include at least one of singlestranded or double stranded DNA or RNA (including mRNA), or a RNA-DNAhybrid. In an embodiment, the sequence corresponds to either afull-length or partial gene sequence. In an embodiment, the sequenceincludes a small inhibitory RNA.

In an embodiment, the T CAR cells are utilized in a method of treating asubject afflicted with a disease or disorder related to the IDSA orUDSA. Such method of treatment is described in detail, and includesadministering specific T CAR cells to the subject in a therapeuticallyeffective amount. In an embodiment, the T CAR cells administered withone or more cytokines, including but not limited to IL-2, IL-4, IL-8,IL-6, TGF-beta, IL-10, BAFF, APRIL-, IL-23, IL-17, an Interferon member,or similar cytokine. For example, it has been reported that modified Tcells used in adoptive therapy are less effective if not stimulated withIL-2 to proliferate. See U.S. Pat. No. 6,410,319, which is incorporatedherein by reference. However, sepsis can be induced if levels ofcytokines are too high. See Jacobson and Ritz, Blood, vol. 118, no. 18,2011, which is incorporated herein by reference. Thus, in an embodiment,the method further includes a computer-selected panel of cytokines foradministration before, during, or after administration of the T CARcells to a particular subject. The computer-selected panel of cytokinesincludes one or more cytokines, including the non-limiting examples setforth herein.

In an embodiment, a subject is treated through administration of atherapeutically effective dose of T CAR cells. In an embodiment, thetherapeutically effective dose includes approximately 10⁶, approximately10⁷, approximately 10⁸, approximately 10⁹, approximately 10¹⁰,approximately 10¹¹, approximately 10¹², or any value there between, TCAR cells per square meter of body surface (cells/m²). In an embodiment,one or more cytokines are infused before, during, or subsequent toadministration of the T CAR cells. In an embodiment, the one or morecytokines are administered in an amount of about 10³, about 10⁴, about10⁵, about 10⁶, or any value therebetween, units per kilogram bodyweight. The dosing schedule is determined based on standard adoptive Tcell therapy schedules, modified as needed for efficacy, cytotoxicity,and other parameters for clinical trials.

In an embodiment, the CAR is contained in a plasmid expression vector inproper orientation for expression, and is transfected (byelectroporation, calcium phosphate, or other means) directly into the Tcell. In an embodiment, the CAR is contained in a viral vector and the Tcells are infected with the artificial construct.

In an embodiment, construction of the CAR is conducted, for example, bystandard PCR techniques such as splice overlap. Assembly of each of oneor more of: target receptor sequence, ribosome binding sequence, signalpeptide, variable regions (VL, VH, etc.), a linker region, hinge region,transmembrane domain, and/or zeta chain is conducted with PCR techniquesand primers either commercially available or derived from the sequenceof the target IDSA or UDSA, when necessary. In an embodiment, the finalconstructs are flanked by restriction sites for directional subcloninginto expression vectors.

As described herein, in an embodiment, the CAR is chromosomallyintegrated at the innate T cell receptor alpha locus. In an embodiment,the CAR is chromosomally integrated in other active chromatin regions(for example the CAR can be linked to the innate T cell receptor-likepromoter region, such as the active T cell receptor V alpha or V betaregion). In an embodiment, the T CAR is integrated at the rearrangedsite of the T cell receptor V beta or V alpha promoter in the engineeredcell line. In an embodiment, each T CAR cell line is propagated with thecorresponding artificial antigen presenting cell to generate the memoryT CAR cell phenotype of CD62L+CD45RA−. In an embodiment, the memory TCAR cells are enriched by cell sorting (e.g., by magnetic or antibodycolumn sorting, FACS, or other technique).

In an embodiment, in vitro propagation cell lines are utilized, such asbut not limited to, Jurkat, Daudi, P815, K562, 2c, and others. In anembodiment, cells are propagated by standard cell culture techniques.

In an embodiment, the T CAR cells further include one or more magneticparticles. In an embodiment, the T CAR cells include one or moremagnetic nanoparticles. In an embodiment, one or more magnetic particlesare administered to a tumor or other disease site of a subject prior to,during, or subsequent to administration of one or more T CAR cellsincluding one or more magnetic particles. In this way, the T CAR cellswill be further directed to the disease site by way of the magneticattraction by way of the magnetic particles both in the T CAR cells aswell as at the disease site.

In an embodiment, the T CAR cells further include one or more radiofrequency identification devices (RFIDs).

In an embodiment, as described above, prior to, during, or subsequent toinfusing the subject with the first cohort of cytolytic T cell CARclones, a sample of cells (e.g., from a biopsy) from the subject's tumoror other target tissue is extracted and tested for cytolysis with theselected T cell clones expected to be used for therapy. In this way, therelative cytolysis is determined, allowing for a more accurate dosingrange and schedule for the subject's therapy without causing unduecytotoxicity.

Various embodiments described herein utilize certain conventionaltechniques and descriptions of molecular biology, polymer chemistry,cell biology, biochemistry, immunology, and organic chemistry.

Computer Systems

In an embodiment, one or more computer systems (including one or morecomputing devices) include computer-readable non-transitory media whichprovides methods to one or more users. In an embodiment, the userincludes a human, or another computer. In an embodiment, a systemincludes a processor. In an embodiment, a system includes non-transitorymedium memory coupled with a processor, the non-transitory medium memorystoring a plurality of machine instructions that cause the processor toperform the steps described herein.

In an embodiment, computer software products are employed, that includecomputer readable non-transitory media including computer-executableinstructions for performing certain logic steps of various methodsdescribed herein. In an embodiment, suitable computer readablenon-transitory media include but are not limited to floppy disc,CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetictapes, etc. In an embodiment, the computer executable instructions arewritten in a suitable computer language or combination of severallanguages. In an embodiment, the various computer software products areutilized for various aspects including but not limited to, dataanalysis, management of data, operation of one or more instruments,storage of data, and further comparison over time of stored data withnew or other stored data.

In an embodiment, a computer-readable non-transitory medium includesencoded programming code for analyzing gene expression. In anembodiment, the software includes code for performing the steps of thedetermination described herein, and a computer-readable non-transitorymedium for storing the code.

In an embodiment, the programming code applies one or more ClusteringAnalysis algorithms, including one or more of K-Means, principalcomponent analysis (PCA), self-organizing maps (SOM), or iterativeindependent component analysis (ICA) to data including one or moremeasured signals to identify an optimum number of independent clustersinto which the data may be grouped. In an embodiment, the programmingcode includes code for one or more of: removing background “noise” fromthe data, using Cluster Analysis to cluster data from one or more geneexpression signals into an optimal number (n) of independent groups, ordetermine if there is a positive correlation of expression of one genein a first group is statistically correlated with the expression of asecond gene in a second group (e.g., if positive gene expression of geneX in primary diseased tissue is correlated with the expression of Y genein metastasized diseased tissue). In an embodiment, the programming codeincludes code that compiles data into a form suitable for computeranalysis.

In an embodiment, the differential screening includes removing “noise”or providing for normalization of the data. Thus, while receptors thatbind specifically to a particular target antigen, hybridizationconditions utilized can be adjusted to decrease the possibility ofnon-specific binding (or background noise). Likewise, in the analysissteps, the computer algorithm(s) utilized herein can provide thecomputational analysis such that the background noise is reduced oreliminated altogether. For example, the computational model can analyzeintensity input of data for a comparative analysis in order to determinewith a statistically high degree of certainty, that a particularreceptor is bound to a target antigen with specificity.

For example, cluster analysis primarily includes top-down or bottom-upanalysis. In addition, massive parallel gene expression monitoringsystems and mathematical methods have been developed for utilizationwith nucleic acid gene array technology. See for example, U.S. Pat. No.7,197,400, which is incorporated herein by reference. For example,top-down analysis (or best-fit analysis) typically starts with a givennumber of clusters and proceeds to partition the data into theseclusters. In this way, the algorithm randomly assigns centers to eachcluster and partitions the nearest data into those clusters. Thealgorithm iteratively finds new centers of the clusters by averagingover the data in the cluster and reassigning data to new clusters as thecenters change. This continues until the centers no longer change. Foranother example, bottom-up clustering (or tree clustering) starts bygrouping data at the lowest level and builds larger groups by bringingthe smaller groups together at the next highest level. In this way, datais clustered together by assigning nearest pairs (according to, forexample, information theoretical criteria or regression methods), thealgorithm progresses up to the next level of joining the two nearestgroups from the prior level as one group. Thus, the number and size ofthe cluster depends on the level achieved. In this way, the algorithmcontinues until the analysis of the similarity of the members inside thecluster compared to the difference across clusters indicates cessation.

For another example, SOMS operate as competitive neural networks, andgroup input data into nearest neighbors based on weights of each neuronreceiving input data. The neuron capable of capturing the data resultsin an update toward the data input. Updating the weights results in ashift in recognition of each neuron toward a center of similar data,thus, the number of neurons provides an estimation of the number ofclusters of data.

For another example, principal component analysis includes a stepwiseanalysis that creates a new component axis at each step where variationis observed in the data. Thus, each component explains the varying basesfor the change in the data. In this way, the analysis projects the datainto a new space spanned by the principal components.

For another example, iterative independent component analysis reducesinput data into components and treats each component as statisticallyindependent from the others. See, for example, U.S. Patent App. Pub. No.2006/0074566, which is incorporated herein by reference. In this way,data is clustered that allows for maximum correlation of individualmembers within a group.

In an embodiment, the computer systems described herein include systemsincluding a computer-readable non-transitory medium on which is encodedprogramming code for analyzing gene expression and/or determiningclustering of gene expression, correlation of gene expression, ordetermining a gene expression profile that is utilized as an antigenicprofile for a particular tissue and/or subject. In an embodiment, a genefunction is derived from the information obtained from one or morebiological assays, including gene expression assays.

Thus, in an embodiment, the antigenic profile includes the qualitativeor quantitative characteristics of the antigens present in a particularanalysis group, such as a tissue type or disease studied.

Thus, in an embodiment, a method for immunotherapy of a subjectcomprises determining the qualitative or quantitative differential geneexpression between a non-diseased tissue type from one or more tissuesamples of two or more non-diseased subjects of a population, and adiseased tissue type from one or more tissue samples of two or morediseased first subjects of a population; deducing the Universal SubjectDisease-Specific Antigens pool for the diseased tissue type based on thedetermination of the differential gene expression; engineering aUniversal Subject Disease-Specific Library of multi-specific T cellsbearing Chimeric Antigen Receptors each specifically recognize one ormore members of the Universal Subject Disease-Specific Antigens pool;obtaining at least one diseased tissue sample from a diseased secondsubject to be treated, wherein the diseased tissue sample from thediseased second subject to be treated corresponds to the same tissuetype as the diseased tissue type from one or more tissue samples of twoor more diseased first subjects of a population; analyzing thequalitative or quantitative gene expression of the diseased tissuesample from the diseased second subject to be treated and determiningthe Universal Subject Disease Specific Antigens pool based on theanalysis (thus, examining one or more qualitative or quantitativecharacteristics of gene expression information, as described herein;comparing the Individual Subject's Disease Specific Antigens pool withthe Universal Subject Disease-Specific Antigens pool and determining oneor more antigen members in common to both pools; selecting one or moremulti-specific T cells bearing Chimeric Antigen Receptors from theUniversal Subject Disease-Specific Library effective against thediseased tissue from the diseased second subject to be treated based onthe determination of the one or more antigen members in common to bothpools; administering a therapeutically effective amount of the selectedone or more multi-specific T cells bearing Chimeric Antigen Receptors tothe diseased second subject to be treated.

In an embodiment, the non-diseased tissue type from one or more samplesof two or more subjects of a population, and the diseased tissue typefrom one or more tissue samples of two or more diseased second subjectsof a population are the same or similar tissue types.

In an embodiment, a method for immunotherapy of a subject comprisesobtaining gene sequence information for at least one diseased tissuetype antigen from a diseased first subject; comparing the gene sequenceinformation for at least one diseased tissue type antigen from adiseased first subject with one or more previously identifiednon-diseased tissue type antigens from two or more second subjects,wherein the diseased tissue type from the diseased first subjectcorresponds to the tissue type of the non-diseased tissue type from thetwo or more second subjects; and determining a Universal SubjectDisease-Specific Antigens pool for a diseased tissue type based on thecomparison; engineering a Universal Subject Disease-Specific Library ofmulti-specific T cells bearing Chimeric Antigen Receptors eachspecifically recognize one or more members of the Universal SubjectDisease-Specific Antigens pool. In an embodiment, the method furtherincludes selecting one or more multi-specific T cells bearing ChimericAntigen Receptors from the Universal Subject Disease-Specific Libraryeffective against one or more antigens of the diseased tissue from thediseased first subject to be treated based on the determination of theone or more antigen members in common to both pools. In an embodiment,the method further includes administering a therapeutically effectiveamount of the selected one or more multi-specific T cells bearingChimeric Antigen Receptors to the diseased first subject to be treated.

In an embodiment, a method for immunotherapy of a subject comprisesdetermining the qualitative or quantitative differential gene expressionbetween a non-diseased tissue type from a subject, and the correspondingdiseased tissue type from the subject; deducing the Individual SubjectDisease-Specific Antigens pool based on the determination of thedifferential gene expression; engineering an Individual SubjectDisease-Specific Library of multi-specific T cells bearing ChimericAntigen Receptors each specifically recognize one or more members of theIndividual Subject Disease-Specific Antigens pool; selecting one or moremulti-specific T cells bearing Chimeric Antigen Receptors from theIndividual Subject Disease-Specific Library based on one or morecriteria as being the most effective against the diseased tissue typefrom the subject; administering a therapeutically effective amount ofthe selected one or more multi-specific T cells bearing Chimeric AntigenReceptors to the subject.

For example, in any of the embodiments described herein, the mosteffective T CAR cells for a subject is determined based on severalfactors, including for example, avidity with which the targetdisease-associated antigen binds the T CAR, the expression level of thetarget disease-associated antigen on the target diseased cell(s), anyclustering of gene expression and the resulting T CAR structuralconfigurations (e.g., a T CAR exhibiting receptor specificity for twodifferent epitopes of the same antigen on a single receptor molecule canbe designed in order to have one receptor specificity with increasedavidity for one epitope over the other epitope, depending on the avidityor level of expression of the epitope on the target diseased cell), orany of the qualitative or quantitative characteristics described herein(e.g., genetic or mRNA mutations, abnormal post-translationalmodifications, variant Kd value, methylation/acetylation of nucleicacids, lipid modifications, relative amount of antigens expressed in adiseased tissue versus normal tissue, a cluster expression of severalparticular antigens in a diseased tissue versus normal tissue, theabsence of expression of a normal tissue antigen in a diseased tissue,cell surface density of a single antigen, cell surface concentration ofa single antigen, number of copies of an antigen per cell, etc.).

In an embodiment, the functional avidity of the T CAR is engineered inorder to increase or decrease the responsiveness of the T CAR to thetarget disease-associated antigen. For example, higher avidity T CARsare likely to exhibit broader recognition of a target disease-associatedepitope, compared to a lower avidity T CAR.

In an embodiment, the recognition domain includes the site at which theT cell receptor physically interacts with the epitope. For example, theavidity of a T cell receptor includes the affinity of the particularepitope with the T cell receptor, but also includes the TCR binding timewith the target antigen/MHC complex. At various levels of TCR avidity,calcium mobilization occurs, target cell lysis occurs, or fullactivation with T cell proliferation occurs. Thus, avidity can bemeasured by chromium release assay, based on SD50 values (concentrationof peptide needed to achieve maximal specific lysis).

In an embodiment, the Kd values of the T CAR cell receptor to antigenbinding affinity ranges from about 5.1 to about 6.1 to about 7.1 toabout 8.1 to about 9.1 to about 10.1 to about 11.1 to about 12.1 toabout 13.1 to about 14.1 to about 15.1 to about 16.1 to about 17.1 toabout 18.1 to about 19.1 to about 20.1 nanomolar. In an embodiment, thefunctional avidity of the T CAR cell receptor to antigen ranges fromabout 1.0-4.0 log pg/mL for half the maximal inhibition efficiency. Inan embodiment, the T CAR cell receptor functional avidity includes about2.0, about 2.5, or about 3.0 log pg/mL for half the maximal inhibitionefficiency.

In an embodiment, the T CAR avidity is inversely proportional to theamount of antigen needed to stimulate a response. Thus, the higher thelevel of antigen needed, the lower the avidity of the T CAR, whereas ifthe avidity level of the T CAR is higher, then a lower amount of antigenis needed to stimulate a response. Typically, this means that the higherthe Kd value of the T CAR binding to the antigen, the higher theavidity. Thus, in an embodiment, for low levels of diseased-associatedantigen(s) expressed on the target diseased cells, the avidity of the TCAR can be modulated to increase the receptor affinity or time contactedwith antigen, for overall increased functional avidity. In anotherembodiment, for high levels of antigen present on the target diseasedcell(s), the avidity of the T CAR can be modulated to decrease theoverall functional avidity, for example by adjusting the receptoraffinity or time contacted with antigen, according to standardpractices.

Likewise, for certain embodiments related to various methods describedherein, the disease-associated antigens (whether Universal or IndividualSubject) are further compared with antigens from one or more vaccinatedsubjects. Thus, in one embodiment, a comparison is made between diseasedand non-diseased tissues (whether from Universal or Individual Subject)and secondarily the output from that first comparison is furthercompared with vaccinated tissues (from a population or from anindividual subject). In one embodiment, a comparison is made directlybetween diseased and vaccinated tissues (whether Universal or IndividualSubject) without first comparing with non-diseased non-vaccinatedtissues. In one embodiment, a comparison is made directly betweennon-diseased and vaccinated tissues (whether Universal or IndividualSubject) without first comparing with diseased tissues.

In an embodiment, a method for immunotherapy of a subject comprises:determining the qualitative or quantitative differential gene expressionbetween a non-diseased tissue type from a subject, and the correspondingdiseased tissue type from the subject; deducing the Individual SubjectDisease-Specific Antigens pool based on the determination of thedifferential gene expression; engineering an Individual SubjectDisease-Specific Library of multi-specific T cells bearing ChimericAntigen Receptors each specifically recognize one or more members of theIndividual Subject Disease-Specific Antigens pool; selecting one or moremulti-specific T cells bearing Chimeric Antigen Receptors from theIndividual Subject Disease-Specific Library based on one or morecriteria as being the most effective against the diseased tissue typefrom the subject; administering a therapeutically effective amount ofthe selected one or more multi-specific T cells bearing Chimeric AntigenReceptors to the subject.

In an embodiment, a composition comprises: a Universal SubjectDisease-Specific library of engineered T cells including one or moremulti-specific Chimeric Antigen Receptors each with selected specificityfor two or more epitopes of one or more Universal SubjectDisease-Specific Antigens on a single receptor.

In an embodiment, a composition comprises: a Universal SubjectDisease-Specific library of engineered T cells including one or moremulti-specific Chimeric Antigen Receptor with selected avidity for twoor more epitopes of one or more Universal Subject Disease-SpecificAntigens on a single receptor. A composition comprises: an IndividualSubject Disease-Specific library of engineered T cells including one ormore multi-specific Chimeric Antigen Receptor with selected avidity fortwo or more epitopes of one or more Individual Subject Disease-SpecificAntigens on a single receptor.

In an embodiment, a composition comprises: an Individual SubjectDisease-Specific library of engineered T cells including one or moremulti-specific Chimeric Antigen Receptors each with selected specificityfor two or more epitopes of one or more Individual SubjectDisease-Specific Antigens on a single receptor.

In an embodiment, a system, comprises: one or more input/output deviceshaving a non-transitory signal bearing medium operable to accept atleast one input related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects; accept at leastone input related to gene expression information from one or morediseased tissue antigens from two or more subjects; compare the inputrelated to gene expression information from one or more non-diseasedtissue antigens from two or more subjects with the input related to geneexpression information from the corresponding one or more diseasedtissue antigens from two or more subjects; generate a differentialantigen value based on the comparison; reinitiate accepting at least oneinput related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects, and comparingwith the input related to gene expression information from thecorresponding gene of the one or more diseased tissue antigens until adifferential threshold is satisfied, in an embodiment the differentialthreshold is based on a pre-determined avidity of the T CAR; andgenerate a Universal Subject Disease-Specific Antigen dataset for thedifferential antigen values.

In an embodiment, a system, comprises: one or more input/output deviceshaving a non-transitory signal bearing medium operable to accept atleast one input related to gene expression information from one or morenon-diseased tissue antigens from a diseased subject to be treated;accept at least one input related to gene expression information fromone or more diseased tissue antigens from the diseased subject to betreated; compare the input related to gene expression information fromthe one or more non-diseased tissue antigens from the diseased subjectwith the input related to gene expression information from thecorresponding one or more diseased tissue antigens from the diseasedsubject; generate a differential antigen value based on the comparison;reinitiate accepting at least one input related to gene expressioninformation from one or more non-diseased tissue antigens from thediseased subject, and comparing with the input related to geneexpression information of the corresponding gene of the one or morediseased tissue antigens until a differential threshold is satisfied, inone embodiment the differential threshold is based on a pre-determinedavidity of the T CAR; and generate an Individual SubjectDisease-Specific Antigen dataset for the differential antigen values.

In an embodiment, an article of manufacture, comprises: a structure foraccepting data related to a subject; a structure for receiving at leastone input related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects; a structure forreceiving at least one input related to gene expression information fromone or more diseased tissue antigens from two or more subjects; astructure for comparing the input related to gene expression informationfrom one or more non-diseased tissue antigens from two or more subjectswith the input related to gene expression information from thecorresponding one or more diseased tissue antigens from two or moresubjects; a structure for generating a differential antigen value basedon the comparison; a structure for reinitiating accepting at least oneinput related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects, and comparingwith the input related to gene expression information from thecorresponding gene of the one or more diseased tissue antigens until adifferential threshold is satisfied in one embodiment the differentialthreshold is based on a pre-determined avidity of the T CAR; and astructure for generating a Universal Subject Disease-Specific Antigendataset for the differential antigen values.

In an embodiment, an article of manufacture, comprises: a structure foraccepting data related to a subject; a structure for receiving at leastone input related to gene expression information from one or morenon-diseased tissue antigens from a diseased subject to be treated; astructure for receiving at least one input related to gene expressioninformation from one or more diseased tissue antigens from the diseasedsubject to be treated; a structure for comparing the input related togene expression information from the one or more non-diseased tissueantigens from the diseased subject with the input related to geneexpression information from the corresponding one or more diseasedtissue antigens from the diseased subject; a structure for generating adifferential antigen value based on the comparison; a structure forreinitiating accepting at least one input related to gene expressioninformation from one or more non-diseased tissue antigens from thediseased subject, and comparing with the input related to geneexpression information of the corresponding gene of the one or morediseased tissue antigens until a differential threshold is satisfied inone embodiment the differential threshold is based on a pre-determinedavidity of the T CAR; and a structure for generating an IndividualSubject Disease-Specific Antigen dataset for the differential antigenvalues.

As used herein, in an embodiment, the differential threshold includes avalue, such as a numerical value, and can include a relative value, ofone or more qualitative or quantitative gene expression informationcharacteristics. Such qualitative and quantitative gene expressioninformation characteristics have been described herein. As describedherein, the differential threshold also includes one or more componentsrelated to a predetermined avidity (T CAR affinity, time contacted toantigen, etc.) and in an embodiment the predetermined avidity is basedon the functional avidity of the T CAR.

In an embodiment, a method executed on a computing device, comprisesaccepting data related to a subject; receiving at least one inputrelated to gene expression information from one or more non-diseasedtissue antigens from two or more subjects; receiving at least one inputrelated to gene expression information from one or more diseased tissueantigens from two or more subjects; comparing the input related to geneexpression information from one or more non-diseased tissue antigensfrom two or more subjects with the input related to gene expressioninformation from the corresponding one or more diseased tissue antigensfrom two or more subjects; generating a differential antigen value basedon the comparison; reinitiating accepting at least one input related togene expression information from one or more non-diseased tissueantigens from two or more subjects, and comparing with the input relatedto gene expression information from the corresponding gene of the one ormore diseased tissue antigens until a differential threshold issatisfied in one embodiment the differential threshold is based on apre-determined avidity of the T CAR; and generating a Universal SubjectDisease-Specific Antigen dataset for the differential antigen values.

In an embodiment, a method executed on a computing device, comprisesaccepting data related to a subject; receiving at least one inputrelated to gene expression information from one or more non-diseasedtissue antigens from a diseased subject to be treated; receiving atleast one input related to gene expression information from one or morediseased tissue antigens from the diseased subject to be treated;comparing the input related to gene expression information from the oneor more non-diseased tissue antigens from the diseased subject with theinput related to gene expression information from the corresponding oneor more diseased tissue antigens from the diseased subject; generating adifferential antigen value based on the comparison; reinitiatingaccepting at least one input related to gene expression information fromone or more non-diseased tissue antigens from the diseased subject, andcomparing with the input related to gene expression information of thecorresponding gene of the one or more diseased tissue antigens until adifferential threshold is satisfied in one embodiment the differentialthreshold is based on a pre-determined avidity of the T CAR; andgenerating an Individual Subject Disease-Specific Antigen dataset forthe differential antigen values.

In an embodiment, a system, comprises circuitry configured for acceptingdata related to a subject; circuitry configured for receiving at leastone input related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects; circuitryconfigured for receiving at least one input related to gene expressioninformation from one or more diseased tissue antigens from two or moresubjects; circuitry configured for comparing the input related to geneexpression information from one or more non-diseased tissue antigensfrom two or more subjects with the input related to gene expressioninformation from the corresponding one or more diseased tissue antigensfrom two or more subjects; circuitry configured for generating adifferential antigen value based on the comparison; circuitry configuredfor reinitiating accepting at least one input related to gene expressioninformation from one or more non-diseased tissue antigens from two ormore subjects, and comparing with the input related to gene expressioninformation from the corresponding gene of the one or more diseasedtissue antigens until a differential threshold is satisfied in oneembodiment the differential threshold is based on a pre-determinedavidity of the T CAR; and circuitry configured for generating aUniversal Subject Disease-Specific Antigen dataset for the differentialantigen values.

In a method executed on a computing device, comprises circuitryconfigured for accepting data related to a subject; circuitry configuredfor receiving at least one input related to gene expression informationfrom one or more non-diseased tissue antigens from a diseased subject tobe treated; circuitry configured for receiving at least one inputrelated to gene expression information from one or more diseased tissueantigens from the diseased subject to be treated; circuitry configuredfor comparing the input related to gene expression information from theone or more non-diseased tissue antigens from the diseased subject withthe input related to gene expression information from the correspondingone or more diseased tissue antigens from the diseased subject;circuitry configured for generating a differential antigen value basedon the comparison; circuitry configured for reinitiating accepting atleast one input related to gene expression information from one or morenon-diseased tissue antigens from the diseased subject, and comparingwith the input related to gene expression information of thecorresponding gene of the one or more diseased tissue antigens until adifferential threshold is satisfied in one embodiment the differentialthreshold is based on a pre-determined avidity of the T CAR; andcircuitry configured for generating an Individual SubjectDisease-Specific Antigen dataset for the differential antigen values.

As described herein, Disease-Specific Antigens (whether Universal orIndividual) includes particular antigens that originate from viruses (indiseases caused by viruses), and also includes antigens that are notparticular to any specific histology or tissue but instead are common todisease, such as tumors. In an embodiment, the Disease-AssociatedAntigens are protein structures that contain mutation sin their sequenceor are exhibit aberrant expression on tumor or diseased tissue versusnormal tissue.

For example, some Disease-Specific Antigens include, but are not limitedto tumor-associated antigens such as EBNA-3, E6, E7, carcinoembryonicantigen (CEA), her-2/neu, Muc-1, MART-1, gp100, tyrosinase, p53,beta-catenin, CDK4, alphafetoprotein, ras, and others. For example, someDisease-Specific Antigens include, but are not limited totumor-associated antigens such as O antigens (lipopolysaccharidemolecules from bacterial membranes), H antigens (components offlagella), K antigens (polysaccharides from bacterial capsules), or“clumping factors” (molecules bound to the outer surface causingagglutination. For example, some Disease-Specific Antigens include, butare not limited to, viral envelope proteins or other viral proteins.

As described in FIG. 1A, and described in more detail in the PropheticExamples section herein, construction of a Universal Memory T cell Lineis conducted, for example, by engineering T memory cells (Tcm) at theCre/lox recombination site, as drawn 100, by standard homologousrecombination techniques. The nucleic acid molecule is placed into thehost cell such that the innate T cell receptor constant region alphachain is disrupted, 110, thereby prohibiting innate T cell receptorformation in the host cell. The engineered cells are selected forCD3(−), CD8+ 120.

As described in FIG. 1B, HLA gene expression is optionally altered suchthat innate host cell NK and T cell elimination of the engineered T CARcells is reduced or eliminated. For example, silencing RNA (siRNA orshRNA) are utilized to prevent expression of HLA I and/or HLA II, whileHLA-E is engineered into the vector, as well as selection markers (e.g.,hygromycin, thymidine kinase) 130. Next, the plasmid expression vectoris transfected into the Cre/lox Tcm cell line as described 140 andpositive clones selected 150 based on the selection criteria. Theresulting Tcm cell clones are Universal Recipient Tcm cell lines asdescribed 160.

As described in FIG. 2A, the structure of a multi-specific CAR, forexample, includes at least two scFVs each specific to two differentepitopes of the same antigen, or two different antigens 200. Theartificially engineered multi-specific CAR is utilized for site-specificintegration into the Universal recipient Tcm cell line, as describedherein. For example, at the lox sites of the targeting vector 210, 220with Cre recombinase expression vector 230 and CMV promoter, with DHFRselectable marker 240.

As described in FIG. 2C, in an embodiment, a multi-specific T CARrecognizes two or more different epitopes of one antigen. That is, a TCAR expressed as a single molecule includes multi-specific epitoperecognition. Having recognition of two or more epitopes of a singleantigen provides for a greater likelihood of the T CAR contacting thetarget antigen. Moreover, since the multi-specific epitope recognitionsites are both on a single molecule, there is a greater chance ofphysical contact with the target antigen. Thus, FIG. 2C illustrates adiseased cell (e.g., tumor cell) exhibiting multiple epitopes of aparticular antigen (Epitope 1 and Epitope 2 of Disease-SpecificAntigen 1) that are specifically recognized by the multi-specific T CARexpressed on the T cell (also illustrated is the internal T CARsignaling domain for activation of the T CAR upon binding with thecognate antigens).

As described in FIG. 2D, in an embodiment, a multi-specific T CARrecognizes two different antigens. That is, a T CAR expressed as asingle molecule includes multi-specific antigen recognition. Havingrecognition of two or more different antigens increases the likelihoodof the T CAR contacting the target antigens. Furthermore, since themulti-specific antigen recognition sites are both on a single molecule,there is a great chance of physical contact with the target antigen (vs.having separate receptors for each antigen). Thus, FIG. 2D illustrates adiseased cell (e.g., tumor cell) exhibiting multiple antigens(Disease-Specific Antigen 1 and Disease-Specific Antigen 2) that arespecifically recognized by the multi-specific T CAR expressed on the Tcell (also illustrated is the internal T CAR signaling domain foractivation of the T cell upon binding of the T CAR with the cognateantigens).

As described in FIG. 3, Construction of a polyclonal Tcm library isconstructed, for example, by obtaining the listing of all currentlyidentified human cell surface antigens 300, and screening the surfaceantigens for specific receptor-antigen pairs with scFv genes 310. Theparameters for screening are set according to the desired outcome, andin one embodiment two scFvs that specifically recognize two differentepitopes on a single antigen are selected 310. From this selectionprocess, a database is created with the sequences, and a freezer stockmaintained 310. Next, multi-specific CAR genes are constructed in thelox targeting vector as described herein, for all of the currentlyidentified human cell surface antigens (˜3700), and a database andfreezer stock maintained 320. The multi-specific CAR vectors aretransfected into the recipient Tcm cell line, resulting in Tcm celllines expressing multi-specific CARs, and a database and freezer stockare maintained 330, producing a polyclonal Tcm library with bi-specificrecognition of approximately 3700 human cell surface antigens 340.

As described in FIG. 4, a Universal Subject Disease-Specific library andMethods of Making and Administering are included. For example, aninitial differential screening of gene expression data 400 of normaltissue cell surface proteins (antigenic profile over a population for aparticular tissue type based on gene expression of non-diseased tissuefrom multiple samples and/or from multiple subjects) compared withdiseased tissue antigens (antigenic profile over a population for aparticular diseased tissue based on gene expression of diseased tissuefrom multiple samples and/or from multiple subjects) is utilized for acomputer determination of Population Disease-Specific Antigens (PDSA)based on the differential screening 410. Next, a library is generatedfor the Universal Subject Disease-Specific Library of multi-specific(including bispecific) T CAR cells effective against the PDSA 420. Next,an individual subject with a specific disease is addressed 430. A biopsyor other cell or fluid sample is obtained from the diseased subject andanalysis of the sample is performed 440.

Next, a computer determination of the antigenic profile of theIndividual Subject's Disease Specific Antigens is made based on theanalysis of the Individual Subject's diseased cells 450. Next, acomputer determination of the selection of which multi-specific T CARcells correspond to the Individual Subject's Disease-Specific Antigensis conducted 460.

Finally, the Individual Subject with specific disease is treated with atherapeutically effective dose of T CAR cells specifically selected forthe subject, based on the analysis of the individual subject's diseasedcells in the context of the Universal Subject Disease Specific Library470. In this way, a library can be constructed for any particular tissuetype or disease type. In an embodiment, several libraries areestablished for various stages of disease progression (e.g., metastasis,or chronic disease such as Alzheimer's or multiple sclerosis).

Regarding the computer systems utilized for the computer determinationsof various aspects described herein, FIG. 4 also describes a flow ofinformation in a system including the computer software and hardwareutilized herein. For example, a computer device or system 486 such asappended to the flow chart of FIG. 4, can be employed. As shown, a CPU482 or other computer processor includes, for example, digital logicprocessors capable of processing input, executing algorithms, andgenerating output as needed in response to the inputs received from aninput device (e.g., keyboard, mouse, monitor, internet, scanner, etc.484). Such processors may include a microprocessor, such as ASIC, andmay include or be in communication with media, such as non-transitorymedia, including computer-readable media which stores instructions that,when executed by the processor, cause the processor to perform the stepsdescribed herein. In an embodiment, data is stored or collected bymemory (e.g., RAM) 480 includes for example, memory storage device,system memory, or cache memory, and can be stored on media, for example,CD-ROM, DVD-ROM, or DVD, floppy disc, etc. 481. In an embodiment, an I/Ocontroller 483 is utilized to manage the data communications of thecomputer system. The I/O controller 483 includes any of a variety ofdevices for accepting and processing information from a user (e.g.machine or human; local or remote), and can include for example, modemcards, wireless cards, network interface cards, sound cards, or othertypes of controllers for any of a variety of standard input devices. Inan embodiment, an output controller of the I/O controller 483 includes,for example, a display device for presenting information to a user(e.g., human or machine; remote or local), and any of the variousiterations includes network or other types of remote communication. Inan embodiment, a transmitter 485 and/or receiver 485 to transmit orreceive data from one database to another or from one central dataprocessing facility to one or more user facilities. In an embodiment, atransmitter and/or receiver includes circuitry configured fortransmitting or receiving information, respectively, as well as anycorresponding software. In an embodiment, a transmitter and/or receiverincludes an antenna, Bluetooth™, or other wireless device, as well asany corresponding software.

As described in FIG. 5, an Individual Subject Disease-Specific libraryand Methods of Making and Administering are included. For example, aninitial differential screening of gene expression data 500 of normaltissue cell surface proteins (antigenic profile for a particular tissuetype based on gene expression of non-diseased tissue from an individualsubject) compared with diseased tissue antigens (antigenic profile for aparticular tissue based on gene expression of diseased tissue from anindividual subject) is utilized for a computer determination ofIndividual Subject Disease-Specific Antigens (ISDSA) based on thedifferential screening 510. Next, a library is generated for theIndividual Subject Disease-Specific Library of multi-specific (includingbispecific) T CAR cells reactive to the ISDSA 520. Next, computerdetermination and selection of the T CAR cells most effective for thesubject based on the multi-specificity of particular antigens isconducted 530. Finally, the individual subject is treated with atherapeutically effective dose of T CAR cells 540.

Regarding the computer systems utilized for the computer determinationsof various aspects described herein, in an embodiment, one or more logicdevices are included. In an embodiment, one or more computing devicesare included.

As illustrated in FIG. 5 also describes a flow of information in asystem including the computer software and hardware utilized herein. Forexample, a computer device or system 594 such as appended to the flowchart of FIG. 5, can be employed. As shown, a CPU 570 or other computerprocessor includes, for example, digital logic processors capable ofprocessing input, executing algorithms, and generating output as neededin response to the inputs received from an input device (e.g., keyboard,mouse, monitor, internet, scanner, etc. 590). Such processors mayinclude a microprocessor, such as ASIC, and may include or be incommunication with media, such as non-transitory media, includingcomputer-readable media which stores instructions that, when executed bythe processor, cause the processor to perform the steps describedherein. In an embodiment, data is stored or collected by memory (e.g.,RAM) 550 includes for example, memory storage device, system memory, orcache memory, and can be stored on media, for example, CD-ROM, DVD-ROM,or DVD, floppy disc, etc. 560. In an embodiment, an I/O controller 580is utilized to manage the data communications of the computer system.The I/O controller 580 includes any of a variety of devices foraccepting and processing information from a user (e.g. machine or human;local or remote), and can include for example, modem cards, wirelesscards, network interface cards, sound cards, or other types ofcontrollers for any of a variety of standard input devices. In anembodiment, an output controller of the I/O controller 580 includes, forexample, a display device for presenting information to a user (e.g.,human or machine; remote or local), and any of the various iterationsincludes network or other types of remote communication. In anembodiment, a transmitter 592 and/or receiver 593 to transmit or receivedata from one database to another or from one central data processingfacility to one or more user facilities. In an embodiment, a transmitterand/or receiver includes circuitry configured for transmitting orreceiving information, respectively, as well as any correspondingsoftware. In an embodiment, a transmitter and/or receiver includes anantenna, Bluetooth™, or other wireless device, as well as anycorresponding software.

As described in FIG. 6, in an embodiment, a listing of gene sequences ofall presently identified human cell surface antigens 600 is utilized fora computer determination of antigens specific to a particular tissuetype (optional step) 610. Next, a Universal Subject Disease SpecificAntigen Library of multi-specific T CAR cells reactive to the listing ofgene sequences is generated 620, and an individual subject with aspecific disease is addressed 630. Next, a biopsy or other cell or fluidsample is obtained from the individual subject at the disease site andanalysis of the individual subject's diseased cells is conducted 640.Next, a computer determination of the Individual Subject's DiseaseSpecific Antigens (ISDSA) based on the analysis of the individualsubject's diseased cells 650. Next, computer determination of selectionof multi-specific T CAR cells are conducted that correspond to theIndividual Subject's Disease-Specific Antigens (ISDSA) 660. Finally, theindividual subject with specific disease is treated with atherapeutically effective dose of selected T CAR cells based on theanalysis of the individual subject's disease-specific antigens in lightof the library.

Regarding the computer systems utilized for the computer determinationsof various aspects described herein, FIG. 6 also describes a flow ofinformation in a system including the computer software and hardwareutilized herein. For example, a computer device or system 617 such asappended to the flow chart of FIG. 6, can be employed. As shown, a CPU612 or other computer processor includes, for example, digital logicprocessors capable of processing input, executing algorithms, andgenerating output as needed in response to the inputs received from aninput device (e.g., keyboard, mouse, monitor, internet, scanner, etc.614). Such processors may include a microprocessor, such as ASIC, andmay include or be in communication with media, such as non-transitorymedia, including computer-readable media which stores instructions that,when executed by the processor, cause the processor to perform the stepsdescribed herein. In an embodiment, data is stored or collected bymemory (e.g., RAM) 611 includes for example, memory storage device,system memory, or cache memory, and can be stored on media, for example,CD-ROM, DVD-ROM, or DVD, floppy disc, etc. 618. In an embodiment, an I/Ocontroller 613 is utilized to manage the data communications of thecomputer system. The I/O controller 613 includes any of a variety ofdevices for accepting and processing information from a user (e.g.machine or human; local or remote), and can include for example, modemcards, wireless cards, network interface cards, sound cards, or othertypes of controllers for any of a variety of standard input devices. Inan embodiment, an output controller of the I/O controller 613 includes,for example, a display device for presenting information to a user(e.g., human or machine; remote or local), and any of the variousiterations includes network or other types of remote communication. Inan embodiment, a transmitter 615 and/or receiver 616 to transmit orreceive data from one database to another or from one central dataprocessing facility to one or more user facilities. In an embodiment, atransmitter and/or receiver includes circuitry configured fortransmitting or receiving information, respectively, as well as anycorresponding software. In an embodiment, a transmitter and/or receiverincludes an antenna, Bluetooth™, or other wireless device, as well asany corresponding software.

In an embodiment, the computing system includes, for example, at leastone of a notebook computer, a work station, a personal data device, adesktop computer, a cluster of processors, a cluster of servers, a cloudcomputing center, a mobile telephone, or other computing device.

In an embodiment, a computer or other processing unit is configured toreceive or transmit information relating to receipt of information froma subject by, for example, a USB cable or wireless network. In anembodiment, a computer or other processing unit is configured forreceiving or storing information.

In an embodiment, the computer or other processing unit is configured toallow input or output of additional information or sharing ofinformation, for example, with a subject's personal medical file orpopulation medical health records. That is, in an embodiment, input oroutput of information related to two or more subjects or an individualsubject can be shared with public health databases, or transferred to asubject's specific medical record (including electronic records). In anembodiment, the information shared with public health databases isanonymized such that identifying information related to the subject hasbeen removed, altered, or encrypted.

Prophetic Examples Example 1 Construction of a Universal Recipient TCell Clone for Efficient CAR Gene Integration and T Cell LibraryConstruction

A cloned universal recipient T cell with a targeted recombination sitefor site-specific introduction of chimeric antigen receptor (CAR) genesis constructed from human cytotoxic T lymphocytes (CTL) using a Cre/loxrecombination system. For example, to create a recipient T cell clone,central memory T cells (Tcm) are purified from human peripheral bloodmononuclear cells (PBMNC) by cell sorting using fluorochrome-conjugatedantibodies for CD8 and CD62L (antibodies and cell sorter available fromBD Biosciences, San Jose, Calif.). Methods to purify and propagate Tcmin vitro are described (see e.g., Berger et al., J. Clin. Investigation118: 294-305, 2008 which is incorporated herein by reference). To createa targeted recombination site in the Tcm, they are transfected with atargeting site vector containing DNA sequences homologous to regionsflanking the gene encoding the constant region of the T cell receptoralpha chain (TCR Ca), and recombination sites for the Cre/loxrecombination system. See FIG. 1A.

Methods and vectors using homologous recombination to integrate DNAconstructs at specific chromosomal sites in mammalian cells aredescribed (see e.g., U.S. Pat. No. 5,202,238 issued to Fell Jr. et al.on Apr. 13, 1993 which is incorporated herein by reference). Also, DNAsand methods to create cells with mutant loxP sites that promotesite-specific integration of exogenous genes are described (see e.g.,Araki et al., Comparative analysis of right element mutant lox sites onrecombination efficiency in embryonic stem cells, BMC Biotechnology 10:29, 2010; Araki et al., Targeted integration of DNA using mutant loxsites in embryonic stem cells, Nucleic Acids Research 25: 868-872, 1997;U.S. Pat. No. 6,130,364 issued to Jakobovits et al. on Oct. 10, 2000 andU.S. Pat. No. 5,928,914 issued to Leboulch et al. on Jul. 27, 1999 whichare incorporated herein by reference). Tcm with the targeting sitevector DNA, including the lox recombination site, inserted in the TCR Cαgene are isolated by cell sorting for CD8⁺, CD3^(neg) cells andselecting for drug-resistance (conferred by a drug resistance marker,e.g., neomycin resistance gene (neo) on the targeting DNA construct).See FIG. 1A 120. See e.g., Araki et al., 1997, Ibid. and U.S. PatentApp. Pub. No. 2012/0060230 by Collingwood et al. published on Mar. 8,2012 which are incorporated herein by reference. CD3^(neg) Tcm cellswith a lox recombination site disrupting the TCRα gene are expanded invitro and subjected to additional recombinant DNA modifications toprevent recognition and killing of the universal recipient T cell cloneby host T cells and NK cells.

To create a universal recipient T cell clone suitable for use insubjects with different HLA haplotypes the CD3^(neg) Tcm cells bearing alox recombination site (see above) are genetically modified to preventrecognition by host (i.e., patient) T cells and NK cells. Expression ofHLA Class I and Class II genes is down-regulated to prevent T cellrecognition of the universal Tcm cells. See FIG. 1B 130. For example,short hairpin RNAs (shRNA) which bind to mRNA encoding Class I (HLA-A,-B, -C) and Class II (HLA-DR, -DQ, -DP) and prevent their translationinto protein may be used to down-regulate HLA protein expression.Various expression vectors and shRNA nucleotide sequences to produceshRNAs in T cells that inhibit HLA protein expression can be utilizedwith various embodiments described herein (see e.g., U.S. PatentApplication No. 2007/0036773 by Cooper et al. published on Feb. 15, 2007which is incorporated herein by reference). For example a 58 nucleotideshRNA which inhibits HLA-A, -B and -C alpha chain expression may beencoded in a plasmid vector containing a selectable marker (see e.g.,U.S. Patent Appl. No. 2007/0036773, Ibid.).

To prevent cytolytic attack by NK cells a nonclassical HLA antigen,HLA-E, is expressed on the Tcm cell surface using an expressionconstruct for HLA-E. A DNA construct encoding HLA-E under the control ofa constitutive promoter element and a selection/suicide gene, hygromycinphosphotransferase/thymidine kinase gene (Hy/TK) are described (seee.g., U.S. Patent Application No. 2007/0036773, Ibid.). The Hy/TK genemay be used for positive (Hy) or negative (TK) selection, followed bytransfection of Cre/lox Tcm cell line 140. Thymidine kinase serves as asuicide gene in the Tcm cells in the event they need to be eliminated invivo. Administration of gancyclovir to Tcm cells expressing TK resultsin the production of toxic metabolites which kill the Tcm cells.Universal Tcm cells which are: CD3^(neg), HLA Class I^(neg), HLA ClassII^(neg), resistant to G418 (i.e. neo^(r)), resistant to hygromycin, andexpress HLA-E are selected and cloned 150. See FIG. 1B. The universalrecipient Tcm cells are used to create Tcm expressing CAR genes througha process of site-specific integration, as described in FIG. 1B 160.

Example 2 Efficient Site-Specific Integration of CAR Genes in theUniversal Recipient Tcm Cell Line

As illustrated in FIG. 1 A, 1 B, 2A, and 2B, multi-specific (at leasttwo specific receptor recognition sites on the same CAR) CAR genes areintegrated at the lox recombination site constructed in the universalrecipient Tcm cell clone at the TCR Cα locus on chromosome 14 (see FIGS.1A and 1B). Cultures of the CD3^(neg), HLA^(neg), HLA-E⁺ Tcm cells witha lox recombination site are transfected with a lox targeting vectorwhich contains: lox recombination sites, a multi-specific CAR gene and aselection marker gene, e.g., dihydrofolate reductase (DHFR) whichconfers resistance to a cytotoxic drug, methotrexate (MTX). See FIG. 2B.The genes for CAR and DHFR are controlled by constitutive promoters,e.g., EF-1α and CMV promoters. Universal recipient Tcm cells arecotransfected with a vector which directs transient expression of theCre recombinase 100 (see e.g., U.S. Pat. No. 6,130,364, Ibid.).Cre-mediated recombination between the lox sites in the Tcm cell and thelox targeting vector results in integration of the multi-specific CARgene and DHFR at the TCRα locus on chromosome 14 110. The transfectedTcm cells are selected in MTX-containing medium and selected forexpression of the multi-specific CAR protein using flow cytometry. Forexample, fluorescent antibodies specific for human IgG Fc hinge are usedto detect Tcm cells expressing a CAR gene. Site specific integration inapproximately 16% of the recipient cells may be observed (see e.g.,Araki et al., 1997, Ibid.)

Example 3 Construction of Polyclonal T Cell Library Expressing CARsRecognizing Currently Identified Human Cell Surface Proteins

A library of CAR genes is constructed to specifically recognize each ofall currently identified human cell surface antigens. See FIG. 3. CARsare constructed with single chain antibodies providing specific bindingto cell surface antigens. A comprehensive list of 3,702 humantransmembrane proteins derived from the DNA sequence of the human genome300 (see e.g., Cunha, et al., Proc. Natl. Acad. Sci. USA 106:16752-16757, 2009 which is incorporated herein by reference) is targetedby the CAR library.

Single chain variable region fragments (SCFv) which bind to each cellsurface antigen (based on the known sequences thereof) may be identifiedby screening phage display libraries comprised of human SCFv 310. Forexample, bacteriophage expressing a library of approximately 3×10⁸ SCFvmay be screened for binding to cell surface antigens (see e.g., De Kruifet al., Proc. Natl. Acad. Sci. USA 92: 3938-3942, 1995 and Rader et al.,Current Opinion Biotechnology 8: 503-508, 1997 which are incorporatedherein by reference). Cell surface antigens to screen the SCFv phagelibrary may be made using recombinant DNA methods to synthesize thecorresponding genes and express them in vitro (see e.g., Sambrook andRussell, 2001, Molecular Cloning, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. which is incorporated herein byreference).

Alternatively, antibodies and SCFv recognizing cell surface antigens maybe obtained from an online database of antibodies against human proteintargets (see e.g., Antibodypedia Nature database online at:antibodypedia dot com.) The identification numbers and locations of thebacteriophage-SCFv clones and the corresponding cell surface antigensthey recognize are stored in a searchable computer database.

The SCFv clones are stored as bacteriophage frozen stocks which can beused to amplify the DNA sequences encoding the SCFvs and incorporatethem in a CAR. Methods to amplify DNA sequences and to construct CARgenes are described (see e.g., U.S. Pat. No. 7,354,762 issued to Jensenon Apr. 8, 2008 which is incorporated herein by reference). For example,a gene encoding a CAR may contain: a SCFv, a human IgG1 Fc hinge domain,a CD4 transmembrane domain, and portions from the cytoplasmic domains ofCD28, CD137 and CD3 zeta chain (see e.g., Park et al. Trends inBiotechnology 29: 550-557, 2011 which is incorporated herein byreference).

To create multi-specific CAR genes a single SCFv is replaced by two SCFvin tandem. See FIG. 2A 200. Conventional methods and peptide sequencesto construct multi-specific antigen receptors are used (see e.g., Macket al., Proc. Natl. Acad. Sci. USA 92: 7021-7025, 1995 which isincorporated herein by reference).

A library of CAR genes is created in a lox targeting vector withspecificities for each of all currently identified cell surface antigensusing recombinant DNA methods (see e.g., Sambrook and Russell, Ibid.) Alibrary of multi-specific CAR genes is created by pairing SCFvs specificfor surface antigens. For example, two different scFvs which recognizetwo different epitopes from a single cell surface protein may be pairedto create a multi-specific CAR. See FIG. 2A 200. Alternatively, scFvsrecognizing two different cell surface proteins may be paired toconstruct a multi-specific CAR. A searchable database is created withrecords for each multi-specific CAR vector including: antigenspecificities, corresponding SCFvs and the freezer location of DNAstocks for each lox targeting vector.

A polyclonal Tcm cell library is constructed using the universalrecipient Tcm cells and the CAR gene library created in the loxtargeting vector. See FIG. 2B 210-230, and FIG. 3 330-340. IndividualCAR genes are transfected into aliquots of approximately 10⁶ recipientcells in individual wells of a multiwell plate (50-100 wells) and placedin selective media containing MTX. See FIG. 2B 240. Surviving Tcm cellsare screened using flow cytometry to identify cells expressing CAR ontheir cell surface. For example, fluorescent antibodies recognizing theIgG1 hinge Fc segment on the CARs may be used for flow analysis. Systemsand methods for high throughput mammalian cell transfection andselection are described (see e.g., Muller-Hartmann et al., Expert Opin.Drug Discov. 2: 1453-1465, 2007 which is incorporated herein byreference). A searchable database is created with records for each Tcmline detailing the location of the Tcm cell stock, the multi-specificCAR expressed, the antigens recognized and the SCFv clones employed 330.A polyclonal Tcm library comprised of approximately 5000 Tcm lines isstored as separate frozen stocks for each Tcm line using standardcryogenic methods for mammalian cells 340. Tcm lines expressingmulti-specific CARs specific for currently identified human cell surfaceproteins, viral antigens, and other pathogens may be included in thelibrary.

Example 4 Treatment of a Breast Cancer Patient with Multiple Tcm LinesExpressing Multi-Specific CARs

A human subject that is a patient with breast cancer is treated withmultiple Tcm lines which are selected based upon the gene expressionprofile of the patient's tumor cells. The patient's breast cancer tumoris surgically removed, and frozen sections are analyzed to obtain tumorcells for preparation of RNA. The identity of all genes expressed by thetumor cells (i.e., the transcriptome) is determined. Methods andinstruments to isolate, amplify and determine the identity, i.e.,nucleotide sequence, of all mRNAs expressed by a tumor cell, areidentified (see e.g., Curtis et al., Nature 486: 346-352, 2012 and theTechnical Bulletin: Whole-Genome Expression Analysis . . . availablefrom Illumina, Inc., San Diego, Calif. which are incorporated herein byreference). The tumor cell transcriptome is analyzed to identify anyexpressed genes encoding cell surface proteins. Bioinformatics tools andmethods to identify any of the approximately 3700 known human cellsurface proteins which are expressed by the tumor cells are described(see e.g., Cunha et al., Ibid.).

Tcm lines expressing multi-specific CARs specific for cell surfaceantigens expressed by the patient's tumor cells are located from thesearchable database and expanded. For example, a breast cancer tumorcell expressing the tumor associated antigens: HER-2/neu, MUC-1, andEGFR may be treated with three Tcm lines, expressing CAR specific foreach of the surface antigens. Multi-specific CARs provide increasedefficacy. For example, multi-specific antibodies have been developed fortargeting cells expressing two antigens (see e.g., U.S. Pat. No.5,601,819, and Phuphanich et al., Cancer Immunol. Immunother. 62:125-135, 2013; each of which is incorporated herein by reference). ThreeTcm lines expressing multi-specific CARs recognizing Her2 or MUC1 orEGFR are retrieved from the library and expanded in vitro to obtainapproximately 10⁹ cells of each line. The patient is infused withapproximately 10⁷ cells/kg of each Tcm line.

To treat metastatic cancer cells which may display a different set ofcell surface antigens relative to the primary tumor, the transcriptomeof metastatic tumor cells or circulating tumor cells may be determined.Tcm lines recognizing a new or altered set of surface antigens areselected from the library and administered to the patient. For example,metastatic breast cancer cells may lose expression of cell surfaceantigens (e.g., HLA-C) and gain expression of other antigens (e.g.,CD51, integrin α_(V)) (see e.g., Kischel et al., Neoplasia 10:1014-1020, 2008 which is incorporated herein by reference. Methods tocollect circulating tumor cells and harvest RNA for expression profilingare described (see e.g., Yu et al., J. Cell Biol. 192: 373-382, 2010which is incorporated herein by reference). Tcm clones specific for thecurrent set of surface antigens expressed by the metastatic tumor cellsare selected from the Tcm library, expanded and administered to thepatient.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations are not expressly set forth herein for sakeof clarity.

While particular aspects of the present subject matter described hereinhave been shown and described, it will be apparent to those skilled inthe art that, based upon the teachings herein, changes and modificationsmay be made without departing from the subject matter described hereinand its broader aspects and, therefore, the appended claims are toencompass within their scope all such changes and modifications as arewithin the true spirit and scope of the subject matter described herein.Furthermore, it is to be understood that the invention is defined by theappended claims. It will be understood by those within the art that, ingeneral, terms used herein, and especially in the appended claims (e.g.,bodies of the appended claims) are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.). It will be further understood by those withinthe art that if a specific number of an introduced claim recitation isintended, such an intent will be explicitly recited in the claim, and inthe absence of such recitation no such intent is present. For example,as an aid to understanding, the following appended claims may containusage of the introductory phrases “at least one” and “one or more” tointroduce claim recitations. However, the use of such phrases should notbe construed to imply that the introduction of a claim recitation by theindefinite articles “a” or “an” limits any particular claim containingsuch introduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

Those having skill in the art will recognize that the state of the arthas progressed to the point where there is little distinction leftbetween hardware and software implementations of aspects of systems; theuse of hardware or software is generally (but not always, in that incertain contexts the choice between hardware and software can becomesignificant) a design choice representing cost vs. efficiency tradeoffs.Those having skill in the art will appreciate that there are variousvehicles by which processes and/or systems and/or other technologiesdescribed herein can be effected (e.g., hardware, software, and/orfirmware), and that the preferred vehicle will vary with the context inwhich the processes and/or systems and/or other technologies aredeployed. For example, if an implementer determines that speed andaccuracy are paramount, the implementer may opt for a mainly hardwareand/or firmware vehicle; alternatively, if flexibility is paramount, theimplementer may opt for a mainly software implementation; or, yet againalternatively, the implementer may opt for some combination of hardware,software, and/or firmware. Hence, there are several possible vehicles bywhich the processes and/or devices and/or other technologies describedherein may be effected, none of which is inherently superior to theother in that any vehicle to be utilized is a choice dependent upon thecontext in which the vehicle will be deployed and the specific concerns(e.g., speed, flexibility, or predictability) of the implementer, any ofwhich may vary. Those skilled in the art will recognize that opticalaspects of implementations will typically employ optically-orientedhardware, software, and/or firmware.

The foregoing detailed description has set forth various embodiments ofthe devices and/or processes via the use of block diagrams, flowcharts,and/or examples. Insofar as such block diagrams, flowcharts, and/orexamples contain one or more functions and/or operations, it will beunderstood by those within the art that each function and/or operationwithin such block diagrams, flowcharts, or examples can be implemented,individually and/or collectively, by a wide range of hardware, software,firmware, or virtually any combination thereof. In one embodiment,several portions of the subject matter described herein may beimplemented via Application Specific Integrated Circuits (ASICs), FieldProgrammable Gate Arrays (FPGAs), digital signal processors (DSPs), orother integrated formats. However, those skilled in the art willrecognize that some aspects of the embodiments disclosed herein, inwhole or in part, can be equivalently implemented in integratedcircuits, as one or more computer programs running on one or morecomputers (e.g., as one or more programs running on one or more computersystems), as one or more programs running on one or more processors(e.g., as one or more programs running on one or more microprocessors),as firmware, or as virtually any combination thereof, and that designingthe circuitry and/or writing the code for the software and or firmwarewould be well within the skill of one of skill in the art in light ofthis disclosure. In addition, those skilled in the art will appreciatethat the mechanisms of the subject matter described herein are capableof being distributed as a program product in a variety of forms, andthat an illustrative embodiment of the subject matter described hereinapplies regardless of the particular type of signal-bearing medium usedto actually carry out the distribution. Examples of a signal-bearingmedium include, but are not limited to, the following: a recordable typemedium such as a floppy disk, a hard disk drive, a Compact Disc (CD), aDigital Video Disk (DVD), a digital tape, a computer memory, etc.; and atransmission type medium such as a digital and/or an analogcommunication medium (e.g., a fiber optic cable, a waveguide, a wiredcommunications link, a wireless communication link, etc.).

In a general sense, those skilled in the art will recognize that thevarious embodiments described herein can be implemented, individuallyand/or collectively, by various types of electro-mechanical systemshaving a wide range of electrical components such as hardware, software,firmware, or virtually any combination thereof; and a wide range ofcomponents that may impart mechanical force or motion such as rigidbodies, spring or torsional bodies, hydraulics, and electro-magneticallyactuated devices, or virtually any combination thereof. Consequently, asused herein “electro-mechanical system” includes, but is not limited to,electrical circuitry operably coupled with a transducer (e.g., anactuator, a motor, a piezoelectric crystal, etc.), electrical circuitryhaving at least one discrete electrical circuit, electrical circuitryhaving at least one integrated circuit, electrical circuitry having atleast one application specific integrated circuit, electrical circuitryforming a general purpose computing device configured by a computerprogram (e.g., a general purpose computer configured by a computerprogram which at least partially carries out processes and/or devicesdescribed herein, or a microprocessor configured by a computer programwhich at least partially carries out processes and/or devices describedherein), electrical circuitry forming a memory device (e.g., forms ofrandom access memory), electrical circuitry forming a communicationsdevice (e.g., a modem, communications switch, or optical-electricalequipment), and any non-electrical analog thereto, such as optical orother analogs. Those skilled in the art will also appreciate thatexamples of electro-mechanical systems include but are not limited to avariety of consumer electronics systems, as well as other systems suchas motorized transport systems, factory automation systems, securitysystems, and communication/computing systems. Those skilled in the artwill recognize that electro-mechanical as used herein is not necessarilylimited to a system that has both electrical and mechanical actuationexcept as context may dictate otherwise.

In a general sense, those skilled in the art will recognize that thevarious aspects described herein which can be implemented, individuallyand/or collectively, by a wide range of hardware, software, firmware, orany combination thereof can be viewed as being composed of various typesof “electrical circuitry.” Consequently, as used herein “electricalcircuitry” includes, but is not limited to, electrical circuitry havingat least one discrete electrical circuit, electrical circuitry having atleast one integrated circuit, electrical circuitry having at least oneapplication specific integrated circuit, electrical circuitry forming ageneral purpose computing device configured by a computer program (e.g.,a general purpose computer configured by a computer program which atleast partially carries out processes and/or devices described herein,or a microprocessor configured by a computer program which at leastpartially carries out processes and/or devices described herein),electrical circuitry forming a memory device (e.g., forms of randomaccess memory), and/or electrical circuitry forming a communicationsdevice (e.g., a modem, communications switch, or optical-electricalequipment). Those having skill in the art will recognize that thesubject matter described herein may be implemented in an analog ordigital fashion or some combination thereof.

Those skilled in the art will recognize that it is common within the artto implement devices and/or processes and/or systems in the fashion(s)set forth herein, and thereafter use engineering and/or businesspractices to integrate such implemented devices and/or processes and/orsystems into more comprehensive devices and/or processes and/or systems.That is, at least a portion of the devices and/or processes and/orsystems described herein can be integrated into other devices and/orprocesses and/or systems via a reasonable amount of experimentation.Those having skill in the art will recognize that examples of such otherdevices and/or processes and/or systems might include—as appropriate tocontext and application—all or part of devices and/or processes and/orsystems of (a) an air conveyance (e.g., an airplane, rocket, hovercraft,helicopter, etc.), (b) a ground conveyance (e.g., a car, truck,locomotive, tank, armored personnel carrier, etc.), (c) a building(e.g., a home, warehouse, office, etc.), (d) an appliance (e.g., arefrigerator, a washing machine, a dryer, etc.), (e) a communicationssystem (e.g., a networked system, a telephone system, a voice-over IPsystem, etc.), (f) a business entity (e.g., an Internet Service Provider(ISP) entity such as Comcast Cable, Quest, Southwestern Bell, etc.), or(g) a wired/wireless services entity (e.g., such as Sprint, Cingular,Nextel, etc.), etc.

Those skilled in the art will appreciate that a user may berepresentative of a human user, a robotic user (e.g., computationalentity), and/or substantially any combination thereof (e.g., a user maybe assisted by one or more robotic). In addition, a user as set forthherein, although shown as a single entity may in fact be composed of twoor more entities. Those skilled in the art will appreciate that, ingeneral, the same may be said of “sender” and/or other entity-orientedterms as such terms are used herein.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely exemplary, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected”, or“operably coupled”, to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable”, to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable and/or physically interactingcomponents and/or wirelessly interactable and/or wirelessly interactingcomponents and/or logically interacting and/or logically interactablecomponents.

All publications, patents and patent applications cited herein areincorporated herein by reference. The foregoing specification has beendescribed in relation to certain embodiments thereof, and many detailshave been set forth for purposes of illustration, however, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A composition comprising: a Universal SubjectDisease-Specific library of engineered T cells including one or moremulti-specific Chimeric Antigen Receptor with selected avidity for twoor more epitopes of one or more Universal Subject Disease-SpecificAntigens on a single receptor. 2.-18. (canceled)
 19. A compositioncomprising: an Individual Subject Disease-Specific library of engineeredT cells including one or more multi-specific Chimeric Antigen Receptorwith selected avidity for two or more epitopes of one or more IndividualSubject Disease-Specific Antigens on a single receptor. 20.-34.(canceled)
 35. A method for immunotherapy of a subject comprising:determining the qualitative or quantitative differential gene expressionbetween a non-diseased tissue type from one or more tissue samples oftwo or more non-diseased subjects of a population, and a diseased tissuetype from one or more tissue samples of two or more diseased firstsubjects of a population; deducing the Universal SubjectDisease-Specific Antigens pool for the diseased tissue type based on thedetermination of the differential gene expression; engineering aUniversal Subject Disease-Specific Library of multi-specific T cellsbearing Chimeric Antigen Receptors each specifically recognize one ormore members of the Universal Subject Disease-Specific Antigens poolwith a pre-determined functional avidity; obtaining at least onediseased tissue sample from a diseased second subject to be treated,wherein the diseased tissue sample from the diseased second subject tobe treated corresponds to the same tissue type as the diseased tissuetype from one or more tissue samples of two or more diseased firstsubjects of a population; analyzing the qualitative or quantitative geneexpression of the diseased tissue sample from the diseased secondsubject to be treated and determining the Universal Subject DiseaseSpecific Antigens pool based on the analysis; comparing the IndividualSubject's Disease Specific Antigens pool with the Universal SubjectDisease-Specific Antigens pool and determining one or more antigenmembers in common to both pools; selecting one or more multi-specific Tcells bearing Chimeric Antigen Receptors from the Universal SubjectDisease-Specific Library effective against the diseased tissue from thediseased second subject to be treated based on the determination of theone or more antigen members in common to both pools; administering atherapeutically effective amount of the selected one or moremulti-specific T cells bearing Chimeric Antigen Receptors to thediseased second subject to be treated. 36.-40. (canceled)
 41. A methodfor immunotherapy of a subject comprising: obtaining gene sequenceinformation for at least one diseased tissue type antigen from adiseased first subject; comparing the gene sequence information for atleast one diseased tissue type antigen from a diseased first subjectwith one or more previously identified non-diseased tissue type antigensfrom two or more second subjects, wherein the diseased tissue type fromthe diseased first subject corresponds to the tissue type of thenon-diseased tissue type from the two or more second subjects; anddetermining a Universal Subject Disease-Specific Antigens pool for adiseased tissue type based on the comparison; engineering a UniversalSubject Disease-Specific Library of multi-specific T cells bearingChimeric Antigen Receptors each specifically recognize one or moremembers of the Universal Subject Disease-Specific Antigens pool with apredetermined functional avidity. 42.-45. (canceled)
 46. A method forimmunotherapy of a subject comprising: determining the qualitative orquantitative differential gene expression between a non-diseased tissuetype from a subject, and the corresponding diseased tissue type from thesubject; deducing the Individual Subject Disease-Specific Antigens poolbased on the determination of the differential gene expression;engineering an Individual Subject Disease-Specific Library ofmulti-specific T cells bearing Chimeric Antigen Receptors eachspecifically recognize one or more members of the Individual SubjectDisease-Specific Antigens pool with a predetermined functional avidity;selecting one or more multi-specific T cells bearing Chimeric AntigenReceptors from the Individual Subject Disease-Specific Library based onone or more criteria as being the most effective against the diseasedtissue type from the subject; administering a therapeutically effectiveamount of the selected one or more multi-specific T cells bearingChimeric Antigen Receptors to the subject. 47.-49. (canceled)
 50. Asystem, comprising: one or more input/output devices having anon-transitory signal bearing medium operable to accept at least oneinput related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects; accept at leastone input related to gene expression information from one or morediseased tissue antigens from two or more subjects; compare the inputrelated to gene expression information from one or more non-diseasedtissue antigens from two or more subjects with the input related to geneexpression information from the corresponding one or more diseasedtissue antigens from two or more subjects; generate a differentialantigen value based on the comparison; reinitiate accepting at least oneinput related to gene expression information from one or morenon-diseased tissue antigens from two or more subjects, and comparingwith the input related to gene expression information from thecorresponding gene of the one or more diseased tissue antigens until adifferential threshold is satisfied based on a predetermined functionalavidity; and generate a Universal Subject Disease-Specific Antigendataset for the differential antigen values. 51.-85. (canceled)